RESEARC H Open Access Sodium arsenite and hyperthermia modulate cisplatin-DNA damage responses and enhance platinum accumulation in murine metastatic ovarian cancer xenograft after hyperthermic intraperitoneal chemotherapy (HIPEC) Clarisse S Muenyi 1 , Vanessa A States 1 , Joshua H Masters 1 , Teresa W Fan 1,2,3,4,5,6 , C William Helm 7 and J Christopher States 1,4,5,6* Abstract Background: Epithelial ovarian cancer (EOC) is the leading cause of gynecologic cancer death in the USA. Recurrence rates are high after front-line therapy and most patients eventually die from pl atinum (Pt) - resistant disease. Cisplatin resistance is associated with increased nucleotide excision repair (NER), decreased mismatch repair (MMR) and decreased platinum uptake. The objective of this study is to investigate how a novel combination of sodium arsenite (NaAsO 2 ) and hyperthermia (43°C) affect mechanisms of cisplatin resistance in ovarian cancer. Methods: We established a murine model of metastatic EOC by intraperitoneal injection of A2780/CP70 human ovarian cancer cells into nude mice. We developed a murine hyperthermic intraperitoneal chemotherapy model to treat the mice. Mice with peritoneal metastasis were perfused for 1 h with 3 mg/kg cisplatin ± 26 mg/kg NaAsO 2 at 37 or 43°C. Tumors and tissues were collected at 0 and 24 h after treatment. Results: Western blot analysis of p53 and key NER proteins (ERCC1, XPC and XPA) and MMR protein (MSH2) suggested that cisplatin induced p53, XPC and XPA and suppressed MSH2 consistent with resistant phenotype. Hyperthermia suppressed cisplatin-induced XPC and prevented the induction of XPA by cisplatin, but it had no effect on Pt uptake or retention in tumors. NaAsO 2 prevented XPC induction by cisplatin; it maintained higher levels of MSH2 in tumors and enhanced initial accumulation of Pt in tumors. Combined NaAsO 2 and hyperthermia decreased cisplatin-induced XPC 24 h after perfusion, maintained higher levels of MSH2 in tumo rs and significantly increased initial accumulation of Pt in tumors. ERCC1 levels were generally low except for NaAsO 2 co-treatment with cisplatin. Systemic Pt and arsenic accumulation for all treatment conditions were in the order: kidney > liver = spleen > heart > brain and liver > kidney = spleen > heart > brain respectively. Metal levels generally decreased in systemic tissues within 24 h after treatment. Conclusion: NaAsO 2 and/or hyperthermia have the potential to sensitize tumors to cisplatin by inhibiting NER, maintaining functional MMR and enhancing tumor platinum uptake. Keywords: cisplatin, sodium arsenite, hyperthermia, HIPEC, metastatic human ovarian cancer, p53, XPA, XPC, MSH2, platinum accumulation * Correspondence: jcstates@louisville.edu 1 Department of Pharmacology & Toxicology, University of Louisville, Louisville, KY 40292, USA Full list of author information is available at the end of the article Muenyi et al. Journal of Ovarian Research 2011, 4:9 http://www.ovarianresearch.com/content/4/1/9 © 2011 Muenyi et al; licensee BioMed Central Ltd. This i s an Open Access article distributed under the terms of the Creative Commons Attribution License (http:// creativecommons.org/licenses/by/2.0), which permits unr estricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Epithelial ovarian cancer (EOC) is the leading cause of gynecological cancer death in the U.S. Approximately 22,000 women a re diagnosed annual ly and 15,000 die from the disease [1]. Most women are diagnosed only after peritoneal dissemination has occurred. The stan- dard treatment for patients with EOC is cytoreductive surgery(CRS)followedbyintravenousPt-taxaneche- motherapy [2]. E ven though initially effective, relapse from residual disease and/or drug resis tant cancer reduces the 5-year survival rate to about 20% [3]. Despite research efforts to improve on Pt-based chemotherapy, or to develop new drugs against EOC, most patients still die from metastatic disease. Since metastatic EOC is usually confined in the peritoneal cavity, it makes theore- tical sense to de liver chemotherapy intraperitoneal ly rather than intravenously since higher levels of drug can be delivered to the disease site by that route [4,5]. In response to three large randomized clinical trials showing benefit to incorporating intraperitoneal (IP) delivery in EOC, the National Cancer Institute issued a clinical announcement recommending that patients with small volume disease at the end of frontline surgery be offered the chance of receiving IP chemotherapy [6]. Adding hyperthermia to chemotherapy agents delivered intraper- itoneally (HIPEC) theoretically could improve outcome [7-9]. Cisplatin is a DNA damaging chemotherapeutic used to treat solid tumors including EOC. However, resistance to cisplatin limits clinic al success. Mechanisms of cispla- tin resistance are multi-factorial and include reduced cel- lular drug accumulation, enhanced drug metabolism by glutathionylation and export by multidrug resistance pro- teins, enhanced DNA damage tolerance and DNA repair [10]. Since Pt-containing chemotherapy drugs remain the major weapon against E OC, improving their efficacy could have a great impact on mortality . The combination of hyperthermia with cisplatin has been reported for the treatment of EOC [11]. Hyperthermia is tumoricidal alone [12 ] and has been shown to enhance cispla tin inhi- bition of peritoneal tumor growth by increasing tumor Pt accumulation [13]. Arsenic trioxide (As 2 O 3 ), an F DA approved drug for the treatment of all-trans-retinoic acid-resistant acute promyelocytic leukemia [14] has the potential to sensit ize tumors to cisplatin [15,16]. Combi- nation chemotherapy studies demonstrate that arsenic sensitizes cancer cells to hyperthermia, radiation, cispla- tin, adriamycin, doxorubicin, and etoposide [16-19]. In vitro studies demonstrate that trivalent arsenic (As 3+ administered as arsenic trioxide [As 2 O 3 ,Trisenox ® ]or sodium arsenite [NaAsO 2 ]) induces apoptosis in multiple types of cancer cells incl uding cervical, melanoma , gas- tric, colon, pancreatic, lung, prostate and ovarian cancer cell lines [20-23]. In vivo studies also show that arsenic inhibits the growth of orthotopic metastatic prostate cancer and peritoneal metastatic ovarian cancer [24,25]. The mechanism of arsenic-induced cell death in vitro is suggested to include formation of oxidative DNA damage [26], activation of the Fas pathway [27], inhibition of DNA repair [28,29], and causation of mitotic arrest and induction of apoptosis in the mitotic cells [20,21]. As 3+ has biological effects similar to those of both cispla- tin and hyperthermia. Like cisplatin it is detoxified by glu- tathionylation and exported by multidrug resistant family transport pumps [30,31], suggesting a potential for compe- tition for the detoxification pathway if arsenic and cisplatin are used in combination. This competition might enhance cisplatin accumulation in cells. Like hyperthermia, As 3+ induces stress response proteins and causes mitotic cata- strophe [21]. T hese actions make arsenic a potentially effective agent to augment hyperthermia enhancement of cisplatin-induced cell death. The goal of this study is to determine how sodium arsenite and hyperthermia modulate mechanisms of cis- platin resistance in vivo. We developed murine models of HIPEC treatment and metastatic human EOC to investigate if NaAsO 2 and hyperthermia alter the expression of DNA repair proteins and tumor platinum levels. We show that NaAsO 2 and hyperthermia either as single agents or in combination r everse key DNA repair protein responses to cisplatin responsible for cis- platin resistance and also enhanced tumor Pt uptake suggesting decreased Pt detoxification. Methods Chemicals Cisplatin and sodium arsenite were purchased from Sigma-Aldrich (St. Louis, MO). Stock solutions (cisplatin 1 mg/mL in 1X PBS and NaAsO 2 13 mg/mL in water) were prepared freshly on the day of treatment and filter sterilized (0.22 μm) prior to use. Cells and cell culture Cisplat in-resistant (A2780/CP70) human ovar ian cancer cells were the kind gift of Dr. Eddie Reed. Cells were maintai ned in RPMI 1640 medium containing 10% fetal bovine serum, 100 μg/mL penicillin/streptomycin, 2 mM L-glutamine and 0.2 units/mL insulin. Cells were cultured in an atmosphere of 95% humidity and 5% CO 2 at 37°C. Cells were passaged twice weekly and replated at a density of 1 × 10 6 cells/150 mm dish. Animals Female NCr athymic nude mice (7 - 9 weeks old), were purchased from Taconic (Cambridge City, IN). Animals were kept in a temperature-controlled room on a 12 h Muenyi et al. Journal of Ovarian Research 2011, 4:9 http://www.ovarianresearch.com/content/4/1/9 Page 2 of 11 light-dark schedule. The animals were maintained in cages with paper filter covers under controlled atmo- spheric conditions. Cages, covers, bedding, food, and water were changed and sterilized weekly. Animals were fedautoclavedanimalchowdietandwater.Allproce- dures were performed under sterile conditions. This experiment was a pproved by the Institutional Animal Care and Use Committee of the University of Louisville in an AALAC approved facility in accordance with all regulatory guidelines. Establishment of intraperitoneal metastatic ovarian tumors in mice A2780/CP70 cell suspensi on (1 × 10 6 cells in 500 μLof serum-free RPMI 1640 media) was injected into the peri- toneum of anesthetized mice using an 18-gauge needle. Theneedlewasflushedwith500μL physiological saline. The abdomen of injected animals was massaged to ensure even distribution of cells. By 3 - 4 weeks after injection, the mice had developed multiple small dissemi- nated IP tumors (1 - 7 mm) (Figure 1). Tumors were monitored by microCT scanning in the Brown Cancer Center Small Animal Imaging Facility. Intraperitoneal chemotherapy Tumor-bearing mice were anesthetized with 3% isoflurane in an inhalation chamber and maintained on 1% isoflurane during surgery. Incisions (~0.5 cm) were made on both sides of the lower abdominal wall allowing entry into the peritoneal cavity (Figure 2). Inflow and outflow tubes were inserted into the perito neal cav ity and secured with skin sutures. The tubes were connected to a bag containing 100 mL normal saline with added cisplatin (3 mg/kg body weight (BW)) ± sodium arsenite (26 mg/kg BW) and cefa- zolin (0.01 mg/mL). ( The do se o f cisp latin used for this study was determined from human dose of cisplatin (100 mg/m 2 ) administered intravenously to a 70 kg (body surface area = 1.87 m 2 ) [32] cancer pa tient and sodium arsenite dose was calculated from a single daily dose of Trisenox (0.15 mg/kg/day) administered intravenously to a 70 kg acute promyelocytic leukemia patient. The underly- ing assumption in the calculations is that the drugs are mixed in 2 L saline solution for HIPEC therapy). The sal- ine bag was subme rged in a w ater ba th to maintain the perfusate temperature at either 37 or 43°C. Perfusion was performed at a rate of 3 mL/min for 60 min using a Mas- terflex pump (Cole-Palmer Instrument Co, Cat # 07524- 50). The inflow and outflow temperatures were monitored by thermocouple probes with temperature maintained within 1°C. The core temperature of the animals was mon- itored using an anal temperature probe and maintained using a heating pad and heat lamp. After 60 min perfusion, most of the perfusate in the peritoneum was sucked out using sterile cotton balls with a light abdominal massage. Wounds were sutured closed and animals were injected intraperitoneally with 1 mL physiological saline containing 0.01 mg ketoprofen for pain. Mice were kept in warm cages (single mouse/cage) and monitored for recovery and discomfort. Immediately (0 h) and 24 h after perfusion, mice were euthanized and tumors, kidneys, liver, spleen, heart and brain were dissected and snap frozen in liquid nitrogen and stored at -80°C until use. Western blot analysis Tumors of ~ 3-5 mm in diameter were homogenized in protein lysis solution ( 1 M Tris-HCl pH 7.4, 0.5 M EDTA, 10% sodium dodecyl sulfate, 180 μg/mL phenyl- methylsulphonylfluoride) using a tissue grinder. After removal of debris by centrifugation (45 min, 14,000 x g), total protein concentration in supernatant was deter- mined by bic inchoninic acid (BCA) method according to manufacturer’ s instructions (Pierce, Rockford, IL, Figure 1 Mouse with multiple small intra peritoneal tumors. A. MicroCT scan of tumors in live mouse. B. Direct visualization of tumors at necropsy of mouse. Three tumors are denoted by arrow in panels A and B. Figure 2 Murine hyperthermic intraperitoneal chemotherapy model. A. Drawing of tumor bearing mouse undergoing HIPEC. Depicted are inlet (a) and outlet (b) ports and anal temperature probe (c) to monitor internal temperature of mouse during perfusion. B. Photograph showing perfusion pump (a), temperature monitor (b), flow tubes (c) and heating bath (d). Mice were perfused for 1 h at the rate of 3 mL/min with cisplatin (3 mg/kg) ± NaAsO 2 (26 mg/kg) at 37 or 43°C. Muenyi et al. Journal of Ovarian Research 2011, 4:9 http://www.ovarianresearch.com/content/4/1/9 Page 3 of 11 micro-well plate protocol) [33]. Fifteen μgproteinsam- ples were resolved by SDS-polyacrylamide gel electro- phoresis and electro-transferred to nitrocellulose membranes. Membranes were probed with antibodies to XPA (Neomarkers, MS-650-P1, dilution 1:1000), XPC (Novus, # ab6264, dilution 1:10,000), GAPDH (Sigma, # A 5441, dilution 1:10,000), p53 (DO-1, Cell Signaling Technology, # 9284, dilution 1:1000), MSH2 (Santa Cruz, # SC-494, dilution 1:1000), and ERCC1 (Santa Cruz, # SC-10785, dilution 1:1000). Secondary antibo- dies (rabbit anti-mouse IgG, # 81-6120 or goat anti-rab- bit, # 81-6120, dilutions 1:2500) conjugated to horseradish peroxidase (Zymed Laboratories, Inc. South San Francisco, CA) were bound to primary antibodies and protein bands detected using enhanced chemilumi- nescence (ECL) substrate (Pierce, Rockford, IL). GAPDH was used as the loading control. Films were scanned with a Molecular Dynamics Personal Densit- ometer SI (Molecular Dynamics, Sunnyvale, CA) and analyzed with ImageQuaNT software (Molecular Dynamics) to determine band density. ICP-MS analysis Samples of tumor homogenates were lyphophilized using Heto vacuum centrifuge (ATR, Laurel, MD) and 350 μL concentrated nitric acid was added to each sample. Wet weight of brain, heart, spleen, liver and kidney was recorded and concentrated nitric acid (350 - 500 μL) was added to samples. Samples were predigested overnight, and then 100 μL of each dissolved sample was transferred into 10 mL acid washed microwavable digestion tubes (triplicate for each sample). The samples were micro- wave-digested at 150°C for 10 min using an automated focused beam mic rowave di gestion s ystem (Explorer ™, CEM, Matthews, NC, USA). After digestion, 1.9 mL of 18 Mohm H 2 O containing 10 ppb internal standard (SPEX CertiPrep, Metuchen, NJ) was added into every sample to give final 5% nitric acid and ICP-MS a nalyses was per- formed using Thermo X Series II ICP-MS (Thermo Fisher Scientific, Waltham, MA) at the University of Louisville Center for Regulatory and Environmental Ana- lytical Metabolomics facility. Concentrated nitric acid was processed similarly as blank. Platinum standard (SPEX CertiPrep, Metuchen, NJ) was used to generate a standard curv e. Platinum and arsenic levels in tumors and tissues were expressed as ng metal/mg protein and ng metal/mg wet weight respectively. Results are pre- sented as the means of three ICP-MS determinations for each data point ± SD from 3 individual mice. Immunocytochemistry Cells (1 × 10 5 ) were plated on poly-D-lysine coated coverslips (BD Biosciences) in a 24-well plate and allowed to acclimate for 24 h. Cells were then treated with 40 μM cisplatin for 1 h. After treatment, cells were washed twice with PBS and incubated in drug-free media for 24 h. Cells were fixed in ice-cold acetone for 10 min at room temperature and washed twice with ice cold PBS and samples incubated for 10 min with PBS containing 0.25% Triton X-100 (PBST). Cells were then washed with P BS three t imes for 5 min and i ncubated in 3% hydrogen peroxide for 30 min to quench endo- genous peroxidase. Cells were washed three times with PBS and incubated in 1% BSA in PBST for 30 min to block unspecific binding of the antibodies. Cells were incubated overnight at 4°C in primary antibodies (1:200 dilution in PBST containing 1% BSA). The primary anti- bodies u sed were XPA (Neomarkers, MS-650-P1), XPC (H-300, SantaCruz Biotechnology, # sc-30156), p53 (DO-1, Cell Signaling Technology, # 9284), MSH2 (Santa Cruz, # SC-494) and ERCC1 (Santa Cruz, # SC- 10785). After incubation, the primary antibody solution was decanted and cells were washed three times with PBS for 5 min each wash. Cells were incubated with sec- ondary antibodies (rabbit anti-mouse IgG, # 81-6120 or goat anti-rabbit, # 81- 6120, dilution 1:200 in PBST con- taining 1% BSA) conjugated to horseradish peroxidase (Zymed Laboratories, Inc. South San Francisco , CA) for 1 h at room temperature. Secondary antibody solution was decanted and cells were washed three times with PBS for 5 min. Cells were stained with 3,3’-diaminoben- zidine (DAB) substrate solution by incubating cells in 200 μL premixed DAB solution (mix 30 μL(onedrop) of the DAB liquid chromogen solution to 2 mL of the DAB liquid buffer s olution (Sigma, # D 3939)) for 1 0 min. DAB solution was removed and cells rinsed briefly with PBS. Cells were counterstained with 20% Wright Giemsa solution for 1 min. Coverslips were mounted on microscope slides using a drop of permount mounting medium. Slides were viewed under a Nikon Eclipse E600 Microscope (Frye r Company Inc, Scientific I nstru- ments, Cincinnati, OH 45240) and pictures taken using MetaMorph software (Universal Imaging Corporation). DAB-positive cells were counted per 1000 cells using MetaMorph software. Statistical analysis Statistical analyses were performed using wilcoxon rank sum test with significance set as p < 0.05, n ≥ 3. Results Murine intraperitoneal chemotherapy model Multiple disseminated tumors were established in the peri- toneal cavity of nude mice as described in Materials and Methods. Mice were scanned using microCT scan to determine the location and estimate the size of tumors (Figure 1A). This was confirmed upon necropsy (Figure 1B). Tumor bearing mice were treated by peritoneal lavage Muenyi et al. Journal of Ovarian Research 2011, 4:9 http://www.ovarianresearch.com/content/4/1/9 Page 4 of 11 for 1 h with cisplatin ± sodium arsenite at 37°C (nor- mothermia) or 43°C (hyperthermia) (Figures 2A and 2B) as described in Materials and Methods. During treatment, the required inflow temperature was reached within 2-5 min after the start of perfusion. Inflow, outflow and rectal temperatures were recorded every 15 min and remained stable within 1°C throughout the 60 min perfu- sion (Table 1). Platinum and arsenic accumulation and retention in metastatic tumors We determined Pt and arsenic accumulation in tumors immediately (0 h) and 24 h after perfusion using ICP-MS. Pt and arsenic accumulated in tumors during treatment (0 h) and generally decreased after treatment (24 h), com- pared with the untreated control (Figure 3). Co-treatment with NaAsO 2 and cisplatin at 37°C (CPA/37 ) or 43°C (CPA/43) caused significantly more Pt to accumulate in tumors. By 24 h after perfusion, tumor Pt levels for CPA/ 37 and CPA/43 treatment conditions decreased to levels similar to CP/37. Hyperthermia did not increase tumor Pt levels nor alter Pt retention in tumors 24 h after treatment. More arsenic initially accumulated in tumors when co- treated with cisplatin and NaAsO 2 at 37°C (CPA/37) than with hyperthermia treatment (CPA/43). Arsenic decreased to similar levels at 24 h. Effect of cisplatin, arsenic and hyperthermia on DNA repair protein expression Cisplatin causes bulky DNA damage that is repaired mostly by the nucleotide excision repair system (NER). Cellular response to cisplatin-DNA damage involves the induction of DNA repair proteins to initiate DNA repair [10]. We determined if NaAsO 2 and hyperthermia modu- lated the expression of XPC, a platinum-DNA damage recognition protein in global genome repair (GGR) [34] subpathway of NER, and of ERCC1 and XPA, down- stream NER proteins that have been implicated in cispla- tin resi stance [35]. We also determined the expression of p53, which is involved in the activation of the GGR path- way by transcriptionally act ivating XPC [36]. In addition to NER, decreased mismatch repair (MMR) has be en implicated in cisplatin resistance [37,38]. Thus, we also investigated the expression of MSH2, an important MMR DNA damage recognition protein. Western blot analysis of p53, XPC, XPA, E RCC1 and MSH2 revealed mouse-to-mouse and tumor-to-tumor variabilities (Figure 4A). Some tumors failed to express the protein of interest while others either expressed high, moderate or very low levels of the proteins. We determined band intensities for the expressed proteins by scanning the films using a Molecular Dynamics Personal Densitometer SI (Molecular Dynamics, Sunnyvale, CA) and analyzing bands of interest using ImageQuaNT software (Molecu- lar Dynamics). Each protein value was normalized to its respective GAPDH (loading control) value. Data were further normalized to untreated control (Figure 4B). Tumors that failed to express the protein of interest were not considere d in the densitome try analyses. P53 (Figure 4B, panel a) and XPC (Figure 4B, panel b) were signifi- cantly induced during treatment (0 h) by cisplati n at 37°C (CP/37) or 4 3°C (C P/43 ) and ci splatin pl us arsenite at 43°C (CPA/43). P53 signific antly decreased at 24 h after treatment with CPA/43 (Figure 4B, panel a). X PC decreased at 24 h after perfusion w ith both CP/43 a nd CPA/43 treatments (Figure 4B, panel b). P53 (Figure 4B, panel a) and XPC (Figure 4B, panel b) did not signifi- cantly increase during (0 h) and af ter (24 h) peritoneal lavage with NaAsO 2 and cisplatin co-tre atment at 37°C (CPA/37). XPA (Figure 4B, panel c) was significantly induced during (0 h) and 24 h after perfusion with CP/ 37, CPA/37 and CPA/43 but not with CP/43. ERCC1 remained generally low for all treatment conditions except with CPA/37 (Figure 4B, panel d). The suppres- sion of MSH2 by CP/37 and CP/43 treatments was not seen in tumors co-treated with arsenite (CPA/37, CPA/43) (Figure 4B, panel e). Expression of P53, XPA and MSH2 in ovarian cancer cells Western blot determination of P53, XPC, XPA, ERCC1 and MSH2 in metastatic tumors revealed that some tumors failed to express p53 (6%), XPC (3%), XPA (8%), ERCC1 (40%) and MSH2 (9%). Failure to express these proteins could be an inherent feature of the cells that were used to establish the tumors or due to mutations and alteration of genes during tumor development that could result in lack of pro tein expression. We therefore per- formed immunocyto chemical st udies using A2780/CP70 cells to determine expression of P53, XPA and MSH2 in these cells (Figure 5A). Immunocytochemistry data revealed that 25% of cells do not express p53 as evident by lack of 3,3’-diaminobenzidine (DAB) brown staining and ~3% and 60% of cells did not stain po sitive for XPA and MSH2 respectively (Figure 5B). Full-length western blots for XPC and ERCC1 had several non-specific bands in addition to the band of interest (data not shown) making it impossible to perform immunocytochemistry with speci- ficity for these proteins. Table 1 Inflow, outflow and body temperatures of mouse during intraperitoneal perfusion Inflow Temperature Outflow Temperature Body Temperature 37.4 ±1.1°C 36.4 ± 0.8°C 35.5 ± 1.0°C 43.0 ± 0.7°C 39.7 ± 0.6°C 36.3 ± 2.1°C Mice were perfused for 1 h with cisplatin (CP/37; CP/43) or cisplatin + NaAsO2 (CPA/37; CPA/43) at 37 or 43°C respectively. Inflow, outflow and body temperatures were recorded every 15 min. Data are presented as means ± SD of readings taken from five mice. Muenyi et al. Journal of Ovarian Research 2011, 4:9 http://www.ovarianresearch.com/content/4/1/9 Page 5 of 11 Platinum and arsenic biodistribution in somatic tissues The clinical use of anticancer chemotherapeutic agents is limited by adverse toxicities. For cisplatin, these include toxicity to the kidney, peripheral nerves, liver, heart, bone marrow and brain [39,40]. Clinical use of arsenic is known to cause liver, kidney a nd neurological damage, cardiovascular and gastro-intestinal toxicity, anemia and leucopenia [41-43]. Therefore, we determined cisplatin and arsenic accumulation in mouse tissues including kid- ney, liver, heart, spleen and brain (Figure 6A and 6B). Samples were prepared as described in Methods. During perfusion, platinum accumulated in all tissues examined regardless of the treatment condition, in the order: kid- ney > liver = spleen > heart > brain. At 24 h after perfu- sion, significant decrease of platinum was observed in the kidney for all treatment conditions. The combination treatment (CPA/43) favo red the removal of platinu m from the liver, spleen and heart at 24 h after perfusion. Arsenic also significantly accumulated in all the tissues examined, in the order: liver > kidney = spleen > heart > brain and it significantly decreased in all tissues by 24 h after perfusion. Discussion Although the platinum analogues (cisplatin and carbopla- tin) are at the forefront of combination treatment for EOC, acquired or inherent resistance limits clinical suc- cess. In the current study, we used metastatic EOC xenograftinnudemicetoinvestigatehowNaAsO 2 and hyperthermia modulate response to cisplatin in vivo.We focused on three key m echanisms of cisplatin resistance: enhanced NER, diminished MMR and decreased Pt accu- mulation. Our data suggest that cisplatin induces resis- tant phenotype in metastatic tumors by inducing XPC and XPA and suppressing MSH2. Sodium arsenite alone or combined with hyperthermia inhibits mechanisms of cisplatin resistance by suppressing XPC induction, main- taining higher levels of MSH2 and increasing tumor uptake of cisplatin. Decreased Pt accumulation is an important mechanism of cisplatin resistance. Hyperthermia has been reported to increase both cellular and DNA Pt levelsin vitro.However, in vivo data remains controversial. Los et al used rats bear- ing metastatic colon cancer to show that hyperthermia suppressed tumor growth by increasing platinum accumu- lation in tumors [13]. Zeamari et al used a similar colon cancer xenograft model in rats and reported that hyperthermia did not increase tumor Pt levels [44]. Similar to Zeamari, we observed that hyperthermia does not increase Pt accumulation in tumors. The observed discre- pancies with Los et al could be due to differences in how HIPEC was performed. Los et al injected hyperthermic cis- platin intraperitoneally; whereas we and Zeamari et al per- formed peritoneal lavage similar to what is done clinically. Unlike hyperthermia, we observed that NaAsO 2 at 37 or 43°C increased initial tumor Pt levels. Since arsenic and Figure 3 Inductively Coupled Plasma Mass Spectrometry (ICP-MS) determination of platinum and arsenic in tumors. Mice were perfused for 1 h with cisplatin (CP/37; CP/43) or cisplatin + NaAsO 2 (CPA/37; CPA/43) at 37 or 43°C respectively. Tumors from untreated (UT) and treated mice were harvested at 0 and 24 h after treatment. Tumors were homogenized and samples of the homogenate were analyzed for protein concentration by BCA or digested in nitric acid for ICP-MS analysis for platinum and arsenic. Data are presented as means ± SEM of ≥3 tumors each from different mice. Statistical analysis was performed using wilcoxon rank sum test. P < 0.05, N ≥ 3: # = lower than 0 h partner, ‡ = higher than CP/37 at 0 h and CP/43 at 0 h, ¶ = higher than CPA/43°C at 0 h. Muenyi et al. Journal of Ovarian Research 2011, 4:9 http://www.ovarianresearch.com/content/4/1/9 Page 6 of 11 Figure 4 DNA repair protein expression in tumors. A. Western blot determination of p53, XPC, XPA, ERCC1 and MSH2 in tumors. GAPDH is loading control. B. Densitometry analyses of (a) p53, (b) XPC, (c) XPA, (d) ERCC1 and (e) MSH2 normalized to GAPDH loading control and untreated tumors. Mice were perfused for 1 h with cisplatin (CP/37; CP/43) or cisplatin plus NaAsO 2 (CPA/37; CPA/43) at 37 or 43°C respectively. Tumors from untreated (UT) mice and treated mice were harvested 0 and 24 h after treatment. Protein extracts were prepared from the tumors and 20 μg loaded per lane for SDS-PAGE. Data are presented as means ± SD of ≥5 tumors each from different mice. Statistical analysis was performed using wilcoxon rank sum test. P < 0.05, N ≥ 5. # = compared to 0 h partner, * = compared to UT. Muenyi et al. Journal of Ovarian Research 2011, 4:9 http://www.ovarianresearch.com/content/4/1/9 Page 7 of 11 cisplatin are detoxified by glutathionylation and export by the multidrug resistant family proteins, potential competi- tion for the deto xification/export pathways might have resulted in more Pt accumulating in the tumors when cis- platin is co-administered with sodium arsenite. Cisplatin i s a DNA damaging agent and p53 is impli- cated in platinum-DNA damage response [36]. P53 is frequently mutated in ovarian cancer [45]. The p53 phe- notype of A2780/CP70 cells remains controversial. Some studies have demonstrated that A2780/CP70 cells have non-functional p53 [46,47], while other studies have shown that these cells have wild type p53 [48,49]. Our data indicate that A2780/CP70 cell population is heterogeneous: ~75% of cells express wild type p53 and ~25% are p53 null (Figure 5). I n addition, 6% of the tumors de rived from A2780/CP70 are p53 null (Figure 4A). Our in vitro data also demonstrate the induction of p53 target genes p21CIP1/WAF1, XPC and DDB2 in A2780/CP70 cells (data not shown), which strongly sug- gests that a large fraction of these cells have wild type p53. The observed heterogeneity might have resulted from mutations and alterations that occur during serial propagation of cells in culture leadin g to cell line drift [50]. The observed hete rogeneity may impact response to c hemotherapy and result in treatment failures because p53 wild type and null cells will respond differ- ently to chemotherapy especially DNA damaging agents such as cisplatin. This heterogeneity explains why tar- geting master regulators such as p53 or AKT in cancer cells has not been successful [51,52]. Therefore, c ombi- nation chemotherapy suc h as cisplatin, sodium arsenite and hyperthermia with different mechanisms of action might be more beneficial than using a single drug to tar- get a single protein or pathway. Cisplatin predominantly forms intrastrand DNA cr oss- links that are repaired by the nucleotide excision repair (NER) system. There are two subpathways of NER; tran- scription coupled repair (TCR) which removes damage from actively transcribing DNA and global genome repair (GGR) which r emoves lesio ns fro m the entire genome [53]. These two pathways differ only in the proteins that are involved in damage recognition. In TCR, CSA and CSB along with RNA pol II recognize damage, whereas in GGR, XPC and DDB2 are important for lesion recogni- tion. XPC is actively involved in the recognition and initia- tion of cisplatin-DNA damage repair in GGR [34,54]. Arsenic has been shown to inhibit NER by inhibiting XPC expression [29]. In the current study, we observed that P53 and XPC were induced by cisplatin. However, NaAsO 2 alone or in combination with hyperthermia prevented the induction of p53 and XPC by cisplatin (Figure 4B, panels a and b). Since p53 is known to tran- scriptionally induce XPC [36], our data suggest that NaAsO 2 ± hyperthermia might be inhibiting p53, which in turn might be suppressing XPC induction. Suppression of XPC will potentially sensitize tumors to cisplatin. Our in vitro data suggest that inhibition of XPC using siRNA sen- sitizes ovarian cancer cells to cisplatin (data not shown). Therefore, the suppression of XPC could potentially sensi- tize tumors to cisplatin in a similar fashion. Following DNA damage recognition, downstream DNA repair pro- teins (XPA, RPA, TFIIH complex, ERCC1/XPF and XPG) are recruited to the DNA damage recognition comple xes in both TCR and GGR to remove the damage i n a com- mon pathway. Over-expression of XPA and ERCC1 mRNA has been associated with cisplatin resistance in ovarian cancer [35]. In the current study, cisplatin induced XPA (Figure 4B, panel c) that was suppressed by Figure 5 Immunocytochemical determination of p53, XPA and MSH2 expression in ovarian cancer cells. A. A2780/CP70 cells were treated for 1 h with 40 μM cisplatin. Cells were washed and incubated in drug-free media for 24 h and immunohistochemistry was performed. Representative pictures of cells at 20x magnification for secondary antibody only control (a), p53 (b), XPA (c) and MSH2 (d). B. Plot of 3,3’-diaminobenzidine (DAB)-positive cells. Data are single biological experiment performed in duplicate slides. Four different fields were counted per coverslip. Muenyi et al. Journal of Ovarian Research 2011, 4:9 http://www.ovarianresearch.com/content/4/1/9 Page 8 of 11 hyperthermia co-treatment (Figure 4 panel c). Suppression of XPA might decrease repair of cisplatin-DNA damage. ERCC1 was modestly induced (<1.5 fold) by NaAsO 2 co-treatment with cisplatin at 37°C (CPA37) (Figure 4B, panel d). In addition to the NER pathway, the mismatch repair (MMR) system has been implicated in cisplatin resis- tance [37]. In an effort to repair Pt-DNA damage by the MMR system, a futile MMR occurs leading to cell death [53,55]. Ovarian cancer cells over-expr essing MMR pro- teins are sensitive to cisplatin [55-57]. We report for the firsttimethattumorstreatedwithcisplatinat37°C (CP37) significantly suppressed MSH2 consistent with resistance. The observed suppression of MSH2 by cis- platin was reversed in tumors co-treated with NaAsO 2 at 3 7 or 43°C ( CPA/37 and CPA/43 respectively) Thus, NaAsO 2 at 37 or 43°C has the potential to sensitize tumors to cisplatin by maintaining functional MMR. Cisplatin causes serious and dose-limiting side effects including kidney da mage, peripheral sensory neuropathy, cardiovascular toxicity, myelosuppression and anemia which occur as a result of diffusion of chemotherapy from the peritoneal to systemic compartment. In addi- tion, arsenic also causes adverse side effec ts including cardiovascular toxicity, kidney damage, myelosuppression and anemia, liver damage and peripheral sensory neuro- pathy. Understanding the biodistribution of these drugs during peritoneal perfusion of chemothe rapy is i mpor- tant in order to predict the occurrence of these adverse side effects and determine the risk:benefit balance in per- forming intraperitoneal perfusion with cisplatin and arsenic. For this reason, we determined platinum and arsenic accumulation in the brain, heart, liver, kidney and spleen during (0 h) and 24 h after perfusion. We observed that platinum and arsenic accumulated to simi- lar extent in these tissues regardless of the treatment condition. The greatest accumulation of Pt was observ ed in the kidney, the site of Pt elimination. Likewise, greatest level of arsenic was observed in the liver, the organ for arsenic metabolism and detoxif ication. Even though we did not observe any toxicity with the short-term survival study, accumulation of arsenic and Pt in assayed organs Figure 6 Platinum and arsenic accumulation in somatic tissues.Micewereperfusedfor1hwithcisplatin(CP/37;CP/43)orcisplatin+ NaAsO 2 (CPA/37; CPA/43) at 37 or 43°C respectively. Tissues from untreated (UT) and treated mice were harvested at 0 and 24 h after treatment. Tissue samples were weighed and digested in nitric acid for ICP-MS analysis for platinum (A) and arsenic (B). Data are presented as means ± SD of triplicate samples each from different mice. Statistical analysis was performed using wilcoxon rank sum test. P < 0.05, N = 3. # = compared to 0 h partner. Muenyi et al. Journal of Ovarian Research 2011, 4:9 http://www.ovarianresearch.com/content/4/1/9 Page 9 of 11 suggests that potential adverse side effects such as ence- phalopathy, cardio toxicity, liv er damage, renal damage and myelosuppression/anemia respectively may occur during long-term survival studies. Conclusions NaAsO 2 alone or combined with hyperthermia is most likely t o enhance cisplati n efficacy because of its abilities to impair NER by inhibiting induction of p53 and XPC and to activate MMR by maintaining high l evels of MSH2 and enhancing platinum accumulation in tumors. NaAsO 2 and hyperthermia might not produce added sys- temic toxicity to cisplatin chemotherapy; on the contrary, the combined treatment might help in the clearance of Pt from tissues. Long-term survival studies are required to determine the efficacy of this new combination che- motherapy. The murine HIPEC model may serve as a useful tool to study in vivo mechanisms of platinum resistance and explore ways to sensitize tumors to plati- num chemotherapy. Abbreviations CP: (cisplatin); CP/37: (cisplatin at 37°C) or CP/43 (cisplatin at 43°C); CPA: (cisplatin plus sodium arsenite); CPA37: (cisplatin plus sodium arsenite at 37° C) or CPA/43 (cisplatin plus sodium arsenite at 43°C); ERCC1: (excision repair cross-complementing 1); GGR: (global genome repair); HIPEC: (hyperthermic intraperitoneal chemotherapy); ICP-MS: (inductively coupled plasma mass spectrometry); NaAsO 2 : (sodium arsenite); MSH2: (human mutS homolog 2); NER: (nucleotide excision repair); Pt: (platinum); TCR: (transcription coupled repair); XPA: (xeroderma pigmentosum group A); XPC: (xeroderma pigmentosum group C). Acknowledgements This work was supported in part by the National Institutes of Health Grant P30ES014443 which supported the collection and analysis of data and the National Science Foundation’s Experimental Program to Stimulate Competitive Research Grant EPS-0447479 which provided the ICP-MS instrumentation and personnel support for the analysis of Pt and As reported in the manuscript. Also, the authors thank Dr. Richard Higashi for technical support with ICP-MS analyses and Dr. Huaiyu Zheng of the Brown Cancer Center Small Animal Imaging Facility for technical assistance with microCT scanning of mice. Author details 1 Department of Pharmacology & Toxicology, University of Louisville, Louisville, KY 40292, USA. 2 Department of Chemistry, University of Louisville, Louisville, KY 40292, USA. 3 Center for Regulatory and Environmental Analytical Metabolomics, University of Louisville, Louisville, KY 40292, USA. 4 Center for Genetics & Molecular Medicine, University of Louisville, Louisville, KY 40292, USA. 5 Center for Environmental Genomics & Integrative Biology, University of Louisville, Louisville, KY 40292, USA. 6 James Graham Brown Cancer Center, University of Louisville, Louisville, KY 40292, USA. 7 Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, St. Louis University School of Medicine, St Louis, MO 63117, USA. Authors’ contributions CSM established metastatic tumor model, performed HIPEC and tissue collection, ICP-MS analysis, western blot analysis, immunohistochemical studies and drafted the manuscript. VAS established metastatic tumor model, performed HIPEC and tissue collection, took and drew pictures for figures 1 and 2. JHM established metastatic tumor model, developed murine HIPEC model in collaboration with CWH, performed HIPEC and tissue collection. TWF provided intellectual input with ICP-MS analysis. CWH developed murine HIPEC model, established metastatic tumor model and participated in study design, coordination, data analysis and manuscript editing. JCS developed murine HIPEC model, established metastatic tumor model and participated in study design, coordination, data analysis and manuscript editing. All authors read and approved the final manuscript. Competing interests Dr Helm has previously received speaking honoraria from ThermaSolutions and grant support from ThermaSolutions and Sanofi-Aventis for clinical research into Hyperthermic Intraperitoneal Chemotherapy for the treatment of ovarian carcinoma. 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Bartelink H, Begg AC: Intraperitoneal cisplatin with regional hyperthermia in advanced ovarian cancer: pharmacokinetics and cisplatin-DNA adduct formation in patients and ovarian cancer cell lines.